The Herpesviridae are responsible for debilitating acute and congenital infections; furthermore, some members of this family are also associated with human cancers. Human Herpes Simplex Viruses Types 1 and 2 (HSV-1 and HSV-2) are responsible for primary and recurrent herpetic lesions of the mouth and genital tract as well as more serious and potentially life-threatening infections of the eye and central nervous system. HSV infections are of particular concern in newborns and in immunocompromised patients. The herpesviruses encode a large number of replication proteins that are excellent targets for antiviral therapy; however, the mechanisms by which they function during DNA replication are poorly understood. The experiments described in this proposal will focus on the interactions of these replication proteins with one another, with DNA at the replication fork and with cellular proteins that may play roles in this process. Events leading to the initiation of viral replication at the origins of replication, the formation of a multiprotein complex at the viral replisome, the priming of Okazaki fragments and interactions with cellular factors during HSV DNA replication will be examined using molecular genetic, biochemical, biophysical and cell biological approaches. Although these features of DNA replication are common for all life forms using DNA as their genetic material, HSV is an ideal system to examine them at the molecular level because this system is amenable to both genetic and biochemical manipulation. The molecular and biochemical characterization of HSV DNA replication is expected to facilitate our understanding of analogous processes in other viral and eukaryotic systems as well as to provide novel targets for antiviral therapy. The specific aims of this proposal are: Aim 1. How is DNA replication initiated in HSV-1 infected cells? Aim 2. How are viral and cellular proteins recruited into multiprotein assemblies that make up the replisome? Aim 3. Identify subdomains of HSV replication proteins for the purpose of domain mapping and eventual structural analysis.